1521-009X/42/4/541545$25.

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DRUG METABOLISM AND DISPOSITION
Copyright 2014 by The American Society for Pharmacology and Experimental Therapeutics

http://dx.doi.org/10.1124/dmd.114.057539
Drug Metab Dispos 42:541545, April 2014

Special Section on Transporters in Toxicity and

DiseaseCommentary
The Role of Transporters in Toxicity and Disease
John D. Schuetz, Peter W. Swaan, and Donald J. Tweedie
Department of Pharmaceutical Sciences, St. Jude Childrens Research Hospital, Memphis, Tennessee (J.D.S); Department of
Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (P.W.S); and Department of Drug Metabolism and
Pharmacokinetics, Boehringer Ingelheim, Ridgefield, Connecticut (D.J.T.)
Received February 5, 2014; accepted February 7, 2014

The significance of transporters in the disposition, metabolism, and

elimination of drugs is well recognized. One gap in our knowledge is
a comprehensive understanding of how drug transporters change
functionality (their amount and activity) in response to disease and
how disease and its inevitable pathology change transporter expression. In this issue of Drug Metabolism and Disposition a series of
review and primary research articles are presented to highlight the
importance of transporters in toxicity and disease. Because of the
central role of the liver in drug metabolism, many of the articles in this

theme issue focus on transporters in the liver and how pathology or

alterations in physiology affects transporter expression. The contributing authors have also considered the role of transporters in
drug interactions as well as drug-induced liver injury. Noninvasive
approaches to assessing transporter function in vivo are also described. Several articles highlight important issues in oncology where
toxicity must be balanced against efficacy. In total, this theme issue
will provide a stepping-stone to future studies that will establish a
more comprehensive understanding of transporters in disease.

Introduction

tissue, the redundancy of transporter function, and the fact that many
tissues express both uptake and efflux transporters in the same or
opposing membranes. For instance, for hepatocytes, we need to
consider the possibility that a drug might interact with both canalicular
efflux and basolateral uptake and efflux transporters in the hepatocyte.
Key findings such as digoxin toxicity (Ho and Kim, 2005) and
myopathy associated with high statin plasma levels (Giacomini et al.,
2013) have been linked to alterations in transporter function. These
events were either a function of drug-drug interactions (DDI), such as
inhibition of P-gp for digoxin (Fenner et al., 2009) and inhibition of
organic anion transporting polypeptides (OATPs) by statins (Link
et al., 2008) or reduced transporter activity for statins with allelic
variants of OATPs (Giacomini et al., 2013). The role of transporters
in various diseases (Ho and Kim, 2005) and in response to drugs
and various toxicants (Ho and Kim, 2005; Sweet, 2005) has been
considered. Just as drug metabolizing enzymes reduce the exposure to
xenobiotics through direct clearance and increased excretion, transporters can also function to modulate the intracellular concentrations
of compounds (McBride et al., 2009; Chu et al., 2013), which would
ultimately alter cellular physiology with toxicologic sequelae.
Deconvoluting the specific role(s) of transporters in tissue and plasma
exposure will come from modeling (Shitara et al., 2006; Poirier et al.,
2009; Menochet et al., 2012) and the application of new approaches to
assess in vivo function, such as positron emission tomography
(Sasongko et al., 2005) and mass spectrometry (Deo et al., 2012). In
comparison with drug metabolizing enzymes there are a limited

Transporters facilitate the movement of compounds across biologic

membranes which, in turn, regulate the intracellular concentration of
a transported substance. These transported compounds can be either
drugs (xenobiotics) or endogenous compounds (endobiotics). On
a rudimentary level the types of transporters can be classified into
uptake and efflux carriers. In vitro model systems, under rigorously
defined conditions, have provided a wealth of information about
individual transporter substrate selectivity, affinity, and driving forces.
From these studies, we know that many drug transporters are
multispecific and feature substrate redundancy, resulting in more than
one transporter interacting with a particular substrate (Giacomini et al.,
2010). One possible explanation for the broad substrate specificity is
the presence of a large substrate binding pocket, as is found in
P-glycoprotein (P-gp, aka MDR1 and ABCB1) (Aller et al., 2009).
Ultimately, the combination of in vitro model systems and structural
analysis will provide mechanistic insights into individual transporters.
However, determining the contribution of a single transporter to
a drugs tissue concentration is complicated by multiple factors,
including the expression of the same transporter in more than one

This work was supported by the National Institutes of Health National Institute
of General Medical Sciences [Grant 2R01GM60964]; National Cancer Institute
[Grants P30CA21745 and CA21865]; and by ALSAC.
dx.doi.org/10.1124/dmd.114.057539.

ABBREVIATIONS: ABC, ATP binding cassette; ABCB11, bile salt export pump, BSEP; ABCC2, multidrug resistance protein 2, MRP2; DDI, drugdrug interaction; DILI, drug-induced liver injury; EMA, European Medicines Agency; FDA, U.S. Food and Drug Administration; MRP, multidrug
resistance protein; OATP, organic anion transporting polypeptide; 6b-OHF, 6b-hydroxycortisol; PCFT, proton-coupled folate transporter; P-gp,
P-glycoprotein; RFC, reduced folate carrier; SBE, steroid-binding element.
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ABSTRACT

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Schuetz et al.

number of specific substrates, and inhibitors for many transporters and

alternative approaches to overcome this significant challenge to
experimental paradigms are needed (Brouwer et al., 2013).
Clearly, transporter affinity, spatial distribution, and membrane
localization influence the degree to which individual transporters
impact a drug or endobiotic concentration. These are important issues
which are not specifically addressed in this theme issue, but have
been reviewed elsewhere (VanWert et al., 2010). Instead, we have
attempted to extend this foundation by presenting both primary and
review articles that highlight the complexity of understanding how
transporters impact disease and are, in turn, affected by disease state.
Liver Transporters in Health and Disease

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Bile acids are synthesized from cholesterol in the liver. After

conjugation, they require transporters, ABCB11 (aka bile salt export
pump, BSEP) and ABCC2 (aka multidrug resistance protein 2, MRP2),
to cross the bile canalicular membranes. These secreted bile acids are
efficiently (.95%) reabsorbed from the intestinal lumen, mostly by the
apical sodium-dependent bile acid transporter (ASBT, SLC10A2) in the
terminal ileum (Dawson et al., 2003). The reabsorbed bile acids return
to the liver via the portal circulation and are taken up by the basolateral
uptake transporters of the hepatocyte (enterohepatic recirculation). The
sodium/taurocholate cotransporting polypeptide (NTCP, SLC10A1)
transports the majority (90%) of these bile acids, but OATPs
(SLCO1B) are also involved. Because transporters play a crucial role
in modulating the uptake and export, hence the amount of liver bile acid,
it is important to have a comprehensive overview of the transporters and
major factors (e.g., nuclear receptors) that are crucial to bile formation
and bile acid homeostasis. Integrating this knowledge is important to
provide insight into hepatic inflammation, fibrosis, and cirrhosis. To
address the role of transporters in the etiology of liver disease, in this
issue, Cuperus et al. (2014) provide an overview of the major canalicular
ATP binding cassette (ABC) transporters and their regulation by nuclear
receptors and function in cholestasis.
Elevation in bilirubin is a biomarker for liver damage, but one caveat
is that significant liver damage is required to raise serum bilirubin levels
(Kaplowitz, 2005). The importance of transporters to bilirubin homeostasis is exemplified by the inclusion of ABCC2 and ABCB11, transporters involved in bilirubin and bile acid transport, in the recently issued
DDI guidances from the European Medicines Agency (EMA) (CHMP,
2012) and the U.S. Food and Drug Administration (FDA) (CDER,
2012). In this context, it is important to understand which transporters
have affinity for and transport bilirubin. Variant ABCC2 alleles can
cause Dubin-Johnson syndrome (Dubin and Johnson, 1954), which is
characterized by a mild conjugated hyperbilirubinemia. However, the
exact importance of ABCC2 in hyperbilirubinemia remains difficult to
assign. For example, patients with Dubin-Johnson syndrome have
hyperbilirubinemia due to loss of MRP2 function and MRP3 upregulation, which increases bilirubin efflux into the systemic circulation
(Konig et al., 1999). As outlined by Keppler (2014), besides ABCC2,
OATP (SLC01B) and other uptake transporters contributes to bilirubin
uptake. Bilirubin is also metabolized in humans exclusively by uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1) in the liver to
form bilirubin glucuronides (Kadakol et al., 2000). Alteration of any, or
all, of these proteins can contribute to increases in bilirubin levels. It is
possible that expression of human transporters in mice and rats will
provide insight into the relative role of human transporters in DDI and
in various disease states (van de Steeg et al., 2013).
The FDA recommends studies with ABCB11 to help provide
a mechanistic understanding of cholestasis, and the EMA guidance
suggests a more proactive role in assessing the interaction of

a compound with this transporter. For example, the causal link relating
inhibition of ABCB11 to cholestasis is not always clear. In this issue,
Rodrigues et al. (2014) consider the interaction with ABCB11 and the
proposed association with cholestasis in a more holistic manner,
discussing bile acid enterohepatic recirculation, bile acid clearance, and
the interplay of additional transporters (MRPs, ABCC subfamily) and
metabolic enzymes (sulfotransferases). The regulation of these transporters and enzymes by nuclear hormone receptors as part of a feedback
loop in response to elevations in bile acids is also presented. Rodrigues
et al. (2014) provide interesting perspectives on the possible contributions
of a number of processes. In addition to the well-appreciated aspects of
bile homeostasis, they consider the possible role of kinases involved in
intracellular trafficking of proteins and also enterobacterial metabolism of
bile acids. As the authors note, the complexity of the overall process can
make it challenging to discern the primary mechanism(s) by which
a drug elevates bile acids either by directly impacting bile acid
homeostasis or as a secondary effect as a consequence of hepatotoxicity.
ABCG2 (breast cancer resistance protein, BCRP) is ubiquitously
expressed and capable of exporting a wide array of endobiotic and
xenobiotic compounds. Studies in knockout mice indicate that Abcg2
(Bcrp) does not have a protective role in adaptation to acute cholestasis
(Mennone et al., 2010), suggesting it does not interact with bile acids.
However, findings from in vitro studies are not as clear-cut and have not
ruled out a role for Abcg2 in bile acid transport. A potential explanation is
that ABCG2 activity is influenced by a combination of the membrane
microenvironment and the inherent properties of ABCG2 (Hegedus et al.,
2009b). For example, recent studies have demonstrated that the activity of
some ABC transporters is regulated by membrane cholesterol levels
(Paulusma et al., 2009). Telbisz et al. (2014) extend this by reporting in
this issue that human ABCG2 contains a domain referred to as a steroidbinding element (SBE). ABCG2 apparently requires this domain because
mutations in the SBE abrogate cholesterol stimulation of ABCG2
ATPase. These studies are extended to show that ATPase activity in
ABCG2 harboring mutations in the SBE are refractory to bile acid
inhibition, unlike wild-type ABCG2. These studies suggest that bile
acids, while not being substrates for ABCG2, may impact its function.
A better understanding of the impact of disease on the expression of
transporters is key not only to developing treatments for specific
conditions but also to understanding the increased or decreased potential
for DDIs in the patient population. Animal models that more closely
recapitulate human diseases can provide needed insight into how
disease progression changes transporter function. Two primary articles
presented in this theme issue address this topic. First, Canet et al. (2014)
apply a model of nonalcoholic steatohepatitis (NASH) as a surrogate of
the human disease to characterize the alterations in transporter function.
In these studies, an interesting outcome of this disease model, in
comparison with healthy animals, was both an up-regulation of efflux
transporters and a repression of uptake transporters. The second article
by Merrell et al. (2014) uses an animal model that mimics human
infection with an enteropathogenic Escherichia coli. They use this
model system to elucidate how the toll-like receptor (TLR) pathway and
certain cytokines modulate expression of the transporters and the
metabolizing enzymes regulating liver bile acid concentrations.
An example of pathogen-induced transporter regulation is also
provided in this issue. In Sub-Saharan Africa, an estimated 25% of all
pregnancies are complicated by placental malaria infection (Dellicour
et al., 2010). The article by Cressman et al. (2014) in this issue takes
a unique perspective on the complex interplay of transporter and
enzyme expression during the pregnancy of malaria-infected female
mice. They find that malaria-induced alterations in the expression of
transporters and drug-metabolizing enzymes in maternal and fetal
tissues may alter drug disposition, potentially impacting maternal and

Transporters in Toxicity and Disease

fetal outcomes. This work is especially relevant in light of the drugs
currently used for malaria treatment (e.g., quinine, chloroquine,
halofantrine, artemisinin, etc.), many of which are also substrates for
CYP3A4 and efflux transporters such as P-gp and MRPs.

Transporters and Oncology

factor is that metabolites (a result of phase I or phase II metabolism)

are likely to interact with transporters. Zamek-Gliszczynski et al.
(2014) in this issue discuss the possible role of metabolites in affecting
transporter function, thus altering the efficacy and safety of drugs. The
definition of a major metabolite according to the metabolites
discussion in the International Harmonization Committees Safety
Testing document (ICH, 2010) is 10% of the total drug-related
material in systemic circulation. Whether this level of metabolite, or
the 25% of parent proposed in the DDI regulatory guidance, is
sufficient to contribute to the overall drug related effects has been
questioned (Yu and Tweedie, 2013). Zamek-Gliszczynski et al. (2014)
also propose that excretory metabolites in modifying transporter
function may also need to be taken into consideration. An important
case could be made for drugs and endobiotics that undergo enterohepatic
recirculation, particularly as levels of noncirculating metabolites can be
significant (Li et al., 2014). A trend in the pharmaceutical industry
toward producing more metabolically stable drug candidates to reduce
clearance may in fact, as the authors propose, lead to greater interactions
with drug transporters. A number of case examples are presented to
illustrate their perspective.
Drugs that inhibit multiple transporters have the capability of
increasing the retention of both drug substrates and endobiotics (e.g.,
bile acids), which may increase the probability of tissue damage.
Indeed, impaired bile-acid export could contribute to the development
of cholestatic drug-induced liver injury (DILI) due to the increased
concentrations of liver bile acids producing hepatocellular apoptosis
and/or necrotic death. ABCB11 is the primary bile acid exporter
located in the canalicular membrane of the hepatocyte. Loss-of-function
mutations in ABCB11 cause the disease progressive familial-intrahepatic
cholestasis type 2 (PFIC 2) (Strautnieks et al., 1998). A number of
studies have highlighted the potential contribution of ABCB11
inhibition to DILI; thus, ABCB11 inhibition is considered a risk factor
in DILI. However, the drugs that interact with ABCB11 in vitro do not
always produce cholestasis. Notably, the basolateral ABC transporters
ABCC3 and ABCC4 also transport bile acids (Zelcer et al., 2003a,b).
In this issue, Kock et al., (2014) hypothesize that interaction with the
basolateral exporters (ABCC3 and ABCC4) might further increase the
likelihood of identifying drugs that produce cholestasis. This is
because ABCC3 and ABCC4 have the capability of exporting bile
acids (Zelcer et al., 2003a) but also because ABCC4 is up-regulated
in cholestasis in mouse and humans (Schuetz et al., 2001; Keitel
et al., 2005). Moreover, Abcc4 also protects against acute cholestatic
injury (Mennone et al., 2006). Thus, predicting drug and transporter interactions contributing to cholestasis might be improved
by knowing which drugs interact with ABCB11 and the basolateral
efflux transporters ABCC4 and ABCC3. Using a combination of
in vitro transport assays and an inventory of drugs associated
with cholestasis, the authors determine that drug interactions with
both ABCB11 and ABCC4 might be important parameters to
consider when assessing a drugs cholestatic potential. At the
very least, this study may facilitate the development of additional
preclinical models to enhance more reliable prediction of druginduced hepatotoxicity.
In Vivo Probes to Dissect Transporter Interaction

Transporters and Drug Interactions

Updates to the recent DDI guidances from both the FDA (draft,
CDER, 2012) and the EMA (CHMP, 2012) include a consideration of
the possible contributions of drug metabolites to DDI and toxicity. It is
usually very challenging to discriminate the relative contribution of
parent drug versus metabolite(s) in DDI. One significant complicating

An emerging area is to noninvasively investigate the functional

capability of the uptake and efflux transporters in the liver using both
contrast and fluorescent dyes. The review by Pastor et al. (2014)
highlights how MRI can provide a noninvasive approach to determining
how hepatic concentrations are impacted by influx and efflux membrane
transport systems. Currently, these dyes exploit the OATPs and MRP2

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The impact of transporters in cancer chemotherapy can be viewed

from two perspectives: decreasing host toxicity and increasing tumoricidal activity. To mitigate and understand the factors causing the
untoward toxicities of cancer chemotherapy, a comprehensive knowledge of the transporters in these tissues is needed. This will be
accomplished with systematic studies investigating the drug specificity
of transporters in susceptible host tissues. Such knowledge can
improve therapeutic efficacy. In this theme issue, Sprowl and
Sparreboom (2014) provide important illustrations of how this can
be realized. They tackle the known uptake carriers and highlight
how unexplained drug-induced pathologies can be explained by
tissue-specific expression of transporters, and they highlight some
potential strategies to minimize clinical toxicity due to transporters.
Knowledge of these transporters can account for the unique side
effects of some chemotherapeutic agents.
P-gp was discovered in the mid-1970s in rodent cells, which acquired
reduced sensitivity to anticancer drugs (Juliano and Ling, 1976). In the
past 20 plus years, other ABC transporters have been discovered that
also confer resistance to chemotherapeutic drugs (Gottesman et al.,
2002). However, much effort has been directed toward developing
inhibitors of P-gp as a strategy to overcome tumor drug resistance. This
focus on P-gp is probably not misplaced, considering the number of
compounds that interact with it. In addition, many of the new targeted
therapies (e.g., tyrosine kinase inhibitors) are substrates for P-gp
(Hegedus et al., 2009a). The strategy to increase antitumor efficacy by
inhibiting P-gp has been effective in vitro but has not successfully
translated in vivo. In this issue, Callaghan et al. (2014) account for this
lack of success. In addition, they propose a paradigm shift beyond the
simple P-gp inhibition model and suggest approaches such as
modulating P-gp expression by affecting signaling pathways as an
approach to increasing antitumor efficacy.
Folates are essential for cell growth and tissue development and
must be obtained from exogenous sources because mammals cannot
synthesize these derivatives de novo. Folates take part in essential
biosynthetic pathways that produce the building blocks for DNA and
RNA as well as certain amino acids. Folates are anions at physiologic
pH and do not cross biologic membranes by diffusion alone. The
major folate transporters are the reduced folate carrier (RFC) (SLC19A1)
and the proton-coupled folate transporter (PCFT) (SLC46A1) (Zhao
et al., 2009). RFC is the major membrane transporter of circulating
folates, and all substrates are anions. Antifolates such as methotrexate
are administered intravenously for cancer treatment. Methotrexate is
also used for the treatment of autoimmune/inflammatory diseases, but
administered orally. Methotrexate and other antifolates are good substrates
for RFC and PCFT. Matherly et al. (2014) provide a comprehensive
review of the biology and role of RFC and PCFT in cancer chemotherapy.

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Schuetz et al.

Conclusions
The past 20 plus years have seen an expansion in the number of
transporters of therapeutic and disease relevance. Transporters have
well-known importance in impacting therapeutic outcome (e.g., RFC is
crucial to antifolate therapy for cancer) or affecting oral drug absorption
and brain penetration (e.g., P-gp). We have attempted to highlight some
important and emerging areas where transporters are critical to drug
response, such as how pathology or changes in physiology affect transporter expression and function. These articles underscore the need to
better understand the whole body distribution of drugs. This can be
accomplished by more clearly elucidating the transport processes
responsible for distribution into various organs and also how disease
and/or other xenobiotics modulate transporters. Such studies suggest
the need for further development of human relevant preclinical models
that incorporate not only expression data but also functional analysis
in particular. The latter determinations of in vivo transporter activity
can be achieved through the use and development of additional
noninvasive probes. The use of in vivo probes of transporter function
will also aid in the prediction of DDIs or the identification of
metabolites that interfere with transporter function. However, although
in vivo approaches will be a boon to our knowledge, in vitro studies
are also essential. In vitro transporter studies can further inform us by
judiciously using databases that catalog drug side effects. In
combination, these approaches will allow us to fill in the gaps in
our understanding of how individual and combinations of transporters
affect tissue concentrations.
Acknowledgments
The authors thank Camille Miller for her outstanding assistance.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Schuetz, Swaan,
Tweedie.
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transporters in the liver. This approach can also reveal variation in

transporter function in both normal and injured hepatocytes. However,
there is also a practical, clinically important reason to know which
transporters use the contrast agents and fluorescent dyes. These dyes are
used to assess liver function, and it important to discriminate between
altered transporter activity versus reduced liver function. Furthermore,
a refined knowledge of transporters involved in transporting contrast dyes
will facilitate assessment and identification of potential DDI in vivo.
Another potential DDI concern is renal tubular secretion. In their
article, Imamura et al. (2014) evaluate 6b-hydroxycortisol (6b-OHF)
as a novel endogenous probe substrate of human OAT3 (SLC22A8)
and multidrug and toxin extrusion protein (MATE, SLC47A1), which
also may be a novel tool to evaluate renal DDI. Typically, 6b-OHF
and 4b-cholesterol are used to evaluate hepatic CYP3A4 activity, but
the renal excretion of 6b-OHF can be affected significantly by
inhibition of OAT3. In an elegant series of experiments using specific
transporter inhibitors, they demonstrate that OAT3, but not MATE,
plays a significant role in the urinary clearance of 6b-OF, thus
revealing a barometer of in vivo OAT3 function.

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Address correspondence to: Dr. John D. Schuetz, Department of Pharmaceutical Sciences, St. Jude Childrens Research Hospital, 262 Danny Thomas Place,
MS 313, Memphis, Tennessee 38105. E-mail: john.schuetz@stjude.org

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